Inbreeding depression is associated with emergence of deleterious effects and loss of genetic variability. Widespread use of genotyping technologies and new approaches for identification of runs-of-homozygosity (ROH) provide valuable tools to better understand the effects of inbreeding depression. We have previously demonstrated that inbreeding affects sperm motility patterns in cattle (Dorado et al. 2016 Reprod. Fertil. Dev. 29, 712-720; 10.1071/RD15324), with an increase in individuals presenting a hyperactivated-like motility. In this study, we characterised ROH patterns and performed gene ontology analysis of a large, highly consanguineous cattle population. Thirty-three Retinta bulls [average inbreeding percentage FPED = 16.57% (10.25 to 30.62%)] were genotyped using the Axiom® BOS 1 High-Density SNP Array (Thermo Fisher Scientific, Waltham, MA, USA). The ROH were estimated using CGATOH package and classified upon their length into 5 categories: 1-2, 2-4, 4-8, 8-16, and >16 Mb, which are inversely related to inbreeding events occurring 50 to 3 generations before, respectively. The ROH showed an average length of 3.73 Mb (1.48 to 6.71 Mb). Total FROH was partially explained by the increase in ROH fragments longer than 8 Mb, which is consistent with recent inbreeding events that occurred in this population in the last 6 generations. Additionally, the distribution of ROH varied notably between chromosomes. For instance, >16 Mb runs (very recent inbreeding event) were absent in BTA26 and BTA29, whereas certain loci on BTA7, BTA13, and BTA24 showed >16 Mb runs in 9 animals. To identify candidate biological functions affected by inbreeding, we performed functional analysis of the genome areas covered by ROH >8 Mb (our pedigree data covered 5.87 equivalent complete generations) using the Functional Annotation Clustering tool implemented in DAVID. Candidate regions were defined by occurring in ROH >8 in more than 6 animals and by a distance <1 Mb between adjacent single nucleotide polymorphisms. Eight significant gene clusters (enrichment score >1.30; P < 0.05) were identified, with 2 of these clusters related to sperm motility. One of these clusters (score 1.72) contained 23 genes coding microtubule-related proteins, which are associated with cellular movement structures such as flagella. The other cluster (score 1.42), included 4 genes related to dynein and motile cilium assembly ATPase complexes, associated with movement of eukaryotic flagella. We observed an additional cluster representing an ATP-binding feature, which included 77 genes (score 1.43). These results indicate that inbreeding could affect sperm motility by altering microtubule structure and motility. However, the fact that ROH were unevenly distributed across the genome, even in a highly inbred cattle population, also suggests that different metabolic pathways could be affected in individuals with similar inbreeding values. Therefore, the use of inbreeding coefficients as predictors for sperm quality should be approached carefully.